Methods for sterilizing cross-linked gelatin compositions

ABSTRACT

Disclosed are methods for sterilizing cross-linked gelatin as well as to sterilized cross-linked gelatin. In particular, the methods of this invention employ E-beam sterilization techniques.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority based on U.S. patent application Ser.No. 10/094,633, now U.S. Pat. No. 6,849,232 entitled “METHODS FORSTERILIZING CROSS-LINKED GELATIN COMPOSITIONS” by Inventors Mark Ashby,Eduardo Chi Sing and Richard J. Greff filed on Mar. 12, 2002.

BACKGROUND OF THE INVENTION Field of Invention

This invention is directed to methods for sterilizing cross-linkedgelatin as well as to sterilized cross-linked gelatin compositionspossessing novel properties. In particular, the methods of thisinvention employ E-beam irradiation to sterilize cross-linked gelatin.

REFERENCES

The following patent applications and patents are cited and/orreferenced in this application as superscript numbers:

-   -   ¹ Correll, et al., Proc. Soc. Exp. Biol. N.Y., 58:233 (1945)    -   ² Correll, et al., Surg. Gyn. and Obst., 82:585 (1945)    -   ³ Correll, et al., U.S. Pat. No. 2,465,357, Therapeutic Sponge        and Method of Making, issued Mar. 29, 1949    -   ⁴ Correll, et al., U.S. Pat. No. 2,507,244, Surgical Gelatin        Dusting Powder and Process for Preparing Same, issued May 9,        1950    -   ⁵ Studer, et al., U.S. Pat. No. 2,558,395, Undenatured Gelatin        Hemostatic Sponge Containing Thrombin, issued Jun. 26, 1951    -   ⁶ Sieger, et al., U.S. Pat. No. 2,899,362, Hemostatic Sponges        and Method of Preparing Same, issued Aug. 11, 1959    -   ⁷ Song, et al., U.S. Pat. No. 5,399,361, Collagen-containing        Sponges as Drug Delivery Compositions for Proteins, issued Mar.        21, 1995    -   ⁸ Cragg, et al., U.S. Pat. No. 6,071,301, Device and Method for        Facilitating Hemostasis of a Biopsy Tract, issued Jun. 6, 2000    -   ⁹ Cragg, et al., U.S. Pat. No. 6,086,607, Device and Method for        Facilitating Hemostasis of a Biopsy Tract, issued Jul. 11, 2000    -   ¹⁰ Cragg, et al., U.S. Pat. No. 6,162,192, System and Method for        Facilitating Hemostasis of Blood Vessel Punctures with        Absorbable Sponge, issued Dec. 19, 2000    -   ¹¹ Pawelchak, et al., U.S. Pat. No. 4,292,972, Lyophilized        Hydrocolloid Foam, issued Oct. 6, 1981    -   ¹² Sawyer, U.S. Pat. No. 4,238,480, Method for Preparing an        Improved Hemostatic Agent and Method of Employing the Same,        issued Dec. 9, 1980    -   ¹³ Sawyer, U.S. Pat. No. 4,404,970, Hemostatic Article and        Method for Preparing and Employing the Same, issued Sep. 20,        1983

All of the above patent applications and patents are herein incorporatedby reference in their entirety to the same extent as if each individualpublication, patient application or patent was specifically andindividually indicated to be incorporated by reference in its entirety.

3. State of Art

Cross-linked gelatin, often in the form of gelatin foam, gelatin film orgelatin sponges, has been used as a hemostatic agent since itsdevelopment by Correll in 1945.¹⁻⁴ In addition, medicaments, such asantibiotics, growth factors and thrombus enhancing agents, have beenincorporated into the cross-linked gelatin enhance the in vivoproperties of the composition.⁵⁻⁷

When used as a hemostatic agent, the cross-linked gelatin is placed onor in the body and, accordingly, the composition must be sterilizedbefore use. Conventionally, sterilization of these cross-linked gelatincompositions is conducted at elevated temperatures for prolonged periodsof time, e.g., 130° to 140° C. for 3 hours as described by Correll.³While the resulting cross-linked gelatin composition is sterile, thesterilization process causes chemical reactions within the cross-linkedgelatin (polypeptide) which results in hardening and insolubilization ofthe gelatin. These changes can be correlated with the tensile strengthand fluid (e.g., water, blood, etc.) uptake of the cross-linked gelatincomposition before and after heat sterilization and the heat sterilizedproduct has higher tensile strength and significantly less fluid uptakeas compared to the pre-sterilized product.

One particular use of cross-linked gelatin described in the art is tofacilitate hemostasis of a puncture site such as a puncture woundresulting from catheter insertion or a biopsy needle. When so used, theart describes ejection of a pledget of cross-linked gelatin from asyringe into the puncture site.⁸⁻¹⁰

Critical to the ejection process is the flowability of the pledget fromthe syringe assembly and retention of its structural integrity duringinsertion into the body. Specifically, ejection of the pledget from thesyringe assembly is preferably conducted with, at most, moderatepressure to ensure accurate placement in vivo which relates to theflowability of the cross-linked gelatin. Higher fluid content pledgetsare believed to correlate with enhance flowability and, accordingly, itis desirable to maintain as high a fluid absorbability content in thesterilized pledget as possible.

Likewise, it is critical that the structural integrity of the pledget issubstantially maintained as it is ejected from the syringe assembly whenplaced in vivo in order to ensure that portions of he pledget are nottorn or otherwise separated from the pledget. This criticality isparticularly important when placed over a blood vessel puncture in orderto avoid unintended thrombosis of the vessel. Structural integrity ofthe gelatin composition of the pledget under pressure is believed tocorrelate with the tensile strength of the composition and, accordingly,it is desirable to maintain as high a tensile strength in the sterilizedpledget as possible.

However, as demonstrated in the Examples below, the heat sterilizationprocesses of the prior art significantly reduce the water absorbabilityof the cross-linked gelatin and only modestly increase its tensilestrength. In addition, heat sterilized gelatin has a significantdrawback when packaged within a device for use because dry heatsterilization requires prolonged heating at elevated temperatures (e.g.,140° C. for 8 hours for a metal component to be sterilized).Notwithstanding such prolonged heating, these processes are oftenineffective in reducing bioburden to a level recognized to effectsterility. The possibility of ethylene oxide or gamma (γ) sterilizationof gelatin has also been mentioned in the literature.¹¹⁻¹³ However,these methods cause irreversible reactions within the gelatin leading toaltered and possibly undersirable physical and biological properties.

As is apparent, methods for sterilizing cross-linked gelatincompositions without significant reductions in fluid absorbability whilesignificantly increasing its tensile strength would be of great value.In addition, it would be particularly valuable if such methods wouldsterilize packaged gelatin compositions, such as those contained withinfinished medical devices such as a delivery system of syringes,syringes, or other assemblies in order to facilitate manufacture ofsterile devices.

SUMMARY OF THE INVENTION

This invention is directed to methods for sterilizing cross-linkedgelatin compositions. In particular, this invention is directed to thenovel and unexpected result that, under carefully controlled conditions,cross-linked gelatin compositions, including packaged gelatincompositions, can be sterilized using E-beam irradiation at roomtemperature conditions. This invention is further directed to thediscovery that the E-beam cross-linked sterilized gelatin compositionsretain a significantly greater amount of fluid absorbability and tensilestrength as compared to heat sterilized cross-linked gelatincompositions.

In a further aspect, it has been discovered that packaged cross-linkedgelatin composition can be sterilized by employing a sufficientlyenergetic E-beam source coupled with an average bulk density of thematerials comprising the packaging elements of no more than about 0.2g/cm³. When so employed, the E-beam sterilizes the package and thecross-linked gelatin composition therein.

In both embodiments, preferred E-beam dosages are from of about 5 to 50kGray and preferably from about 15 to 25 kGray.

Accordingly, in one of its method aspects, this invention is directed toa method for sterilizing a cross-linked gelatin composition which methodcomprises exposing the cross-linked gelatin composition to a sufficientdose of E-beam irradiation under conditions wherein said composition issterilized.

In another of its method aspects, this invention is directed to a methodfor preparing a sterile, cross-linked gelatin composition in a packagingelement wherein the method comprises:

(a) selecting a packaging element;

(b) adding said cross-linked gelatin composition to the packagingelement selected in (a) above; and

(c) exposing the packaging element formed in (b) above to a sufficientdosage of E-beam irradiation maintained at an initial fluence of atleast 5 μCurie/cm² to sterilize both the packaging element and thecross-linked gelatin composition therein wherein the average bulkdensity of the materials comprising the packaging element is less thanabout 0.2 gm/cm³.

As noted above, the E-beam sterilized cross-linked gelatin compositionsretain a significantly greater amount of fluid absorbability and tensilestrength as compared to heat sterilized cross-linked gelatincompositions. Accordingly, in one of its composition aspects, thisinvention is directed to a sterilized cross-linked gelatin compositioncharacterized by a fluid absorbability of at least 30 grams of fluid pergram of gelatin and a tensile strength of greater than 2.0 lbs/in².

In a preferred embodiment, the sterilized cross-linked gelatincomposition has a fluid absorbability of at least 35 grams of fluid pergram of gelatin and a tensile strength of greater than 2.5 lbs/in².

In another preferred embodiment, the sterilized cross-linked gelatincomposition comprises a medicament such as an antimicrobial agent (e.g.,an antibiotic), growth factors, thrombus enhancing agents, and the likeor a property modifying agent such as a wetting agent. Mixtures ofmedicaments and property modifying agents can also be used. Suitablemedicaments can be mixed with or impregnated into the cross-linkedgelatin composition prior to E-beam sterilization. Incorporation ofbiocompatible wetting agents into a cross-linked gelatin composition istypically conducted prior to E-beam sterilization. Incorporation of suchwetting agents is described in U.S. Provisional Patent Application Ser.No. 60/275,420 entitled “Cross-Linked Gel Compositions Comprising aWetting Agent”, and U.S. Patent Application Ser. No. 10/068,812, alsoentitled “Cross-Linked Gel Compositions Comprising a Wetting Agent”,which applications are incorporated herein by reference in theirentirety.

In still another of its method aspects, this invention is directed to amethod for preparing a sterile syringe assembly comprising across-linked gelatin composition wherein said syringe assembly isincluded in a packaging element which method comprises:

(a) selecting a syringe assembly comprising a holding chamber aninjection port which comprising a luer hub and an ejection port which isattached to a cannula;

(b) adding to the holding chamber of said syringe assembly across-linked gelatin composition in the form of a pledget;

(c) adding sterile saline to said holding chamber wherein sufficientamounts of said saline are added to hydrate said pledget;

(d) transferring the hydrated pledget into the cannula attached to theejection port;

(e) combining at least a single syringe produced in (d) above into apackaging element;

(f) exposing the packaging element formed in (e) above to a sufficientdosage of E-beam irradiation maintained at an initial fluence of atleast 5 μCurie/cm² to sterilize the packaging element and the syringeand the gelatin composition therein

wherein the average bulk density of the materials comprising thepackaging element is less than about 0.2 gm/cm³.

Preferably, the packaging element is a poly-Tyvec packaging such as thatwhich available from Mangar Corp., City of Industry, Calif., USA.

Preferably, the initial fluence of E-beam radiation is preferably at 10μCurie/cm² and, more preferably, at least 5 μCurie/cm².

More preferably, each packaging element comprises from 1 to about 20syringes and even more preferably from 1 to 2 syringes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is directed to methods for sterilizing cross-linkedgelatin as well as to sterilized cross-linked gelatin. Prior todescribing this invention in further detail, the following terms willfirst be defined.

Definitions

As used herein, the following terms have the following meanings:

The term “cross-linked gelatin” refers to well known gelatin foams,films or sponges which are cross-linked with a conventionalcross-linking agent such as formaldehyde as described in the art byCorrell.¹⁻³ The term “cross-linked gelatin composition” refers tocompositions comprising cross-linked gelatin. Such compositions ofteninclude other components such as a medicament⁸⁻¹⁰ or a second polymersuch as collagen¹³ or starch.⁶

The term “growth factors” refer to those medicaments which areconventionally employed to facilitate tissue growth such as theendothelial wall of a punctured blood vessel. Examples of suitablegrowth factors include PDGF, EGF, FGF, IGF, TGF, and the like.

The term “thrombus enhancing agents” refer to those medicaments whichare conventionally employed to facilitate thrombus formation at apuncture site such as at the endothelial wall of a punctured bloodvessel. Examples of suitable thrombus enhancing agents include thrombin,fibrinogen, factor XIII, and other coagulation factors.

The term “antimicrobial agent” refers to agents which destroy microbes(i.e., bacteria, fungi, viruses and microbial spores) thereby preventingtheir development and pathogenic action. Preferred antimicrobial agentsinclude antibiotics and antiviral agents and, in particular,antibiotics.

The term “initial fluence” of E-beam radiation refers to the fluence ofthis beam immediately after release from the E-beam accelerator. As iswell known, the fluence of an E-beam will be reduced the further ittravels from the source.

The term “packaging element” refers to those packaging components usedto encase the cross-linked gelatin and include, by way of example,boxes, syringes, envelops, tubings, catheters, introducers and the like.The packaging elements may comprise glass, plastic, paper, ceramics,cardboard, and the like.

The term “average bulk density” refers to the weight of total product tobe sterilized divided by its volume.

The term “syringe assembly” refers to the syringe body comprising anopen proximal end which acts as the addition port of said syringe, anopen distal end which acts as the ejection port of said syringe, acavity between said proximal and distal ends which acts as a holdingchamber for said syringe, and a plunger which, when activated, acts toeject material residing in the holding chamber out of the distal end ofthe syringe assembly.

Methods

The methods of this invention involve E-beam sterilization of across-linked gelatin composition which method comprises exposing thecross-linked gelatin composition to a sufficient dose of E-beamirradiation under conditions wherein said composition is sterilized.

In a preferred embodiment, the cross-linked gelatin composition to besterilized comprises a medicament such as an antimicrobial agent (e.g.,an antibiotic), growth factors, thrombus enhancing agents, and the likeor a property modifying agent such as a wetting agent. Mixtures ofmedicaments and property modifying agents can also be used. Suitablemedicaments can be mixed with or impregnated into the cross-linkedgelatin composition prior to E-beam sterilization. Incorporation ofbiocompatible wetting agents into a cross-linked gelatin composition istypically conducted prior to E-beam sterilization. Incorporation of suchwetting agents is described in U.S. Provisional Patent Application Ser.No. 60/275,420 entitled “Cross-Linked Gel Compositions Comprising aWetting Agent”, and U.S. Patent Application Ser. No. 10/068,812, alsoentitled “Cross-Linked Gel Compositions Comprising a Wetting Agent”,which applications are incorporated herein by reference in theirentirety.

When employed, the medicament is utilized in an amount sufficient forits intended purpose, e.g., an antimicrobially effective amount, anamount sufficient to induce thrombus formation, an amount sufficient topromote growth. The specific amount employed relates to theeffectiveness of the medicament, the disease condition of the patientbeing treated, the age and weight of the patient, the location of thedisease and other factors well within the purview of the attendingclinician.

Also when employed, the wetting agent is utilized in an amountsufficient to decrease the time to fully hydrate the composition.Preferably, the cross-linked gelatin composition will comprise fromabout 0.001 to about 20 weight percent of the wetting agent based on thetotal weight of the composition. Preferably, the composition comprisesfrom about 0.005 to about 10 weight percent.

The dose of E-beam radiation employed is one sufficient to sterilize thecross-linked gelatin composition. In a preferred embodiment, the E-beamdosage is preferably from about 5 to 50 kGray and more preferably fromabout 15 to about 25 kGray with the specific dosage being selectedrelative to the quantity of cross-linked gelatin composition to besterilized as well as the amount of bioburden estimated to be thereon.Such factors are well within the skill of the art. Upon completion ofthe sterilization process, the sterilized product is ready for shipmentto the ultimate user.

E-beam sterilization is preferably conducted at ambient atmosphericconditions such as a temperature of from about 15° C. to about 30° C.and the exposure time of the product to the E-beam radiation isdependent on the fluence of the radiation employed and the dosagerequired which is well within the skill of the art. Preferably, exposuretime of the product to the E-beam is less than 5 minutes and, morepreferably, from about 1 to about 180 seconds.

Preferably, the composition is exposed to E-beam irradiation having aninitial fluence of at least 5 μCurie/cm² and, more preferably, from atleast 5 μCurie/cm² to at least 15 μCurie/cm² and an E-beam dosage arefrom of about 5 to 50 kGray and more preferably from about 15 to 25kGray.

The E-beam sterilized cross-linked gelatin compositions retain asignificantly greater amount of fluid absorbability and tensile strengthas compared to heat sterilized cross-linked gelatin compositions. Forexample, the E-beam sterilized cross-linked gelatin compositiondescribed herein preferably have a fluid absorbability of at least 30grams of fluid per gram of gelatin and a tensile strength of greaterthan 2.0 lbs/in².

In a more preferred embodiment, the sterilized cross-linked gelatincomposition has a fluid absorbability of at least 35 grams of fluid pergram of gelatin and a tensile strength of greater than 2.5 lbs/in².

In another embodiment, the E-beam sterilization methods of thisinvention can be used to sterilize a packaging element comprising across-linked gelatin composition. When so employed, it is necessary toensure that the packaging element comprising the cross-linked gelatincomposition is exposed to a sufficient dosage of E-beam irradiationmaintained at an initial fluence of at least 5 μCurie/cm² to sterilizethe packaging element and its contents. Because of the low penetratingcapacity of the E-beams, the average bulk density of the materialscomprising the packaging element should be less than about 0.2 gm/cm³.

In one embodiment, the packaging element comprises the syringe and, inanother, the syringe is further packaged into a box or other suitablecontainer. In the latter embodiment, the average bulk density of thepackaging element is measured relative to the syringe and the containerand the container contain from 1 to about 20 syringes but preferablyfrom 1 to 2 syringes.

Utility

The methods of this invention are useful in providing sterilizedcross-linked gelatin compositions which can then be used as hemostaticagen with mammals and, in particular, humans.

The following example illustrates certain embodiments of the inventionbut are not meant to limit the scope of the claims in any way.

EXAMPLE

In the example below, all temperatures are in degrees celsius (unlessotherwise indicated) and all percents are weight percent (also unlessotherwise indicated). Additionally, the following abbreviations have thefollowing meanings. If an abbreviation is not defined, it has itsgenerally accepted meaning.

cm² = square centimeter g = gram kGy or kGray = kiloGray lbs = poundsμCuries = microcurie USP = US Pharmacopia

Example Sterilization of Cross-Linked Gelatin Compositions

Three gelatin compositions were cross-linked with approximately 0.01equivalents of formaldehyde. The first sample (Sample A) was used asunsterilized control, the second sample (Sample B) was E-beam sterilizedby E-beams for a period of about 30 seconds to provide for a dose ofabout 17-25 kGy; and the third sample (Sample C) was heat sterilized byexposure of the sample to a temperature of approximately 130° C. forabout 3 hours.

The color, tensile strength and fluid uptake properties of each of thesamples was then determined and are repeated below:

Color Tensile Strength Water Uptake Sample A White 1.5 lbs/in² 42 g/gSample B White 2.7 lbs/in² 36 g/g Sample C Off White 2.0 lbs/in² 28 g/g

The color was determined visually. Water uptake was measured per the USPtest for absorbable gelatin. Tensile strength was determinedconventionally using a Chatillion gauge.

The above example demonstrates that E-beam irradiation of cross-linkedgelatin provides for sterilized compositions having significantly highertensile strength and water uptake properties as compared to heatsterilized cross-linked gelatin.

From the foregoing description, various modifications and changes in thecomposition and method will occur to those skilled in the art. All suchmodifications coming within the scope of the appended claims areintended to be included therein.

1. A method for preparing a sterile, cross-linked gelatin compositionfor use in vivo, comprising: providing a cross-linked gelatincomposition; exposing the cross-linked gelatin composition to a dose ofE-beam irradiation, the dose of E-beam irradiation being sufficient tosterilize the cross-linked gelatin composition, and the dose of E-beamirradiation being sufficient to increase the tensile strength of thecross-linked gelatin composition to a tensile strength of at least about2.0 lbs/in².
 2. The method of claim 1 wherein the composition after theirradiation has a water absorbability of at least about 30 grams ofwater per gram of the composition.
 3. The method of claim 1 wherein thecomposition further comprises an antimicrobial agent, a growth factor, athrombus enhancing agent, or mixtures thereof.
 4. The method of claim 3wherein the dose of the irradiation is from about 17 to about 25 kGy. 5.The method of claim 1, in which the dose of E-beam irradiation issufficient to increase the tensile strength of the cross-linked gelatincomposition by more than approximately 80%.
 6. A method for preparing asterile, cross-linked gelatin composition for use in vivo, comprising:providing a cross-linked gelatin composition; exposing the cross-linkedgelatin composition to a dose of E-beam irradiation, the dose of E-beamirradiation being sufficient to sterilize the cross-linked gelatincomposition, the dose of E-beam irradiation being sufficient to retainno less than approximately 85% of the water absorbability of thecross-linked gelatin composition, and the dose of E-beam irradiationbeing sufficient to increase the tensile strength of the composition toa tensile strength of at least about 2.0 lbs/in².
 7. The method of claim6 wherein the composition after the irradiation has a waterabsorbability of at least about 30 grams of water per gram of thecomposition.
 8. The method of claim 6 wherein the composition furthercomprises an antimicrobial agent, a growth factor, a thrombus enhancingagent, or mixtures thereof.
 9. The method of claim 8 wherein the dose ofthe irradiation is from about 17 to about 25 kGy.
 10. A method forpreparing a package of sterile, cross-linked gelatin composition for usein vivo, comprising: providing a packaging element; providing across-linked gelatin composition; enclosing the cross-linked gelatincomposition in the packaging element; and exposing the packaging elementto a dose of E-beam irradiation, the dose of E-beam irradiation beingsufficient to sterilize the entire package, and the dose of E-beamirradiation being sufficient to increase the tensile strength of thecross-linked gelatin composition to a tensile strength of at least about2.0 lbs/in².
 11. The method of claim 10 wherein the composition afterthe irradiation has a water absorbability of at least about 30 grams ofwater per gram of the composition.
 12. The method of claim 10 whereinthe composition further comprises an antimicrobial agent, a growthfactor, a thrombus enhancing agent, or mixtures thereof.
 13. The methodof claim 12 wherein the dose of the irradiation is from about 17 toabout 25 kGy.
 14. The method of claim 10, in which the dose of E-beamirradiation is sufficient to increase the tensile strength of thecross-linked gelatin composition by more than approximately 80%.